Prevalence, bacterial causes, and antimicrobial susceptibility profile of mastitis isolates from cows in large-scale dairy farms of Northern Ethiopia

The study was undertaken to determine the prevalence of bovine mastitis, isolate mastitis causing bacteria, assess the association of some risk factors, and determine the antibiotic resistance pattern of bacterial isolates in cows in large-scale dairy farms of Northern Ethiopia. A total of 305 lactating and nonlactating cows were included in the present study. The overall prevalence of clinical and subclinical mastitis was 3.6 and 33.8?%, respectively. The quarter level prevalence was 15.4?%; from which, 11.9 and 1.1?% were subclinical form and blind teat, respectively, while the remaining 2.4?% were of clinical form. Staphylococcus aureus accounted for 36?% of the isolates followed by Escherichia coli (27.3?%). Risk factors including age (p?9?years (OR?=?3.63, 95?% CI?=?1.42-9.25)), cows with four to seven calves (OR?=?3.39, 95?% CI?=?2.06-5.60), and cows in late lactation stage (OR?=?3.79, 95?% CI?=?1.64-8.75). In multivariable logistic regression analysis, age (p?=?0.005) and lactation stage (p?=?0.027) showed statistically significant association with the occurrence of mastitis. The antimicrobial susceptibility pattern showed high susceptibility of S. aureus to nalidixic acid (82.4?%) followed by chloramphenicol (58.8?%); however, these species were resistant to the rest of the antimicrobials tested. Highest resistance was observed against clindamycin and ampicillin. Coliform bacteria (E. coli and Klebsiella pneumoniae) showed resistance to most of the antimicrobials used. Detailed investigation is needed to identify the interplay of managemental and environmental risk factors to design appropriate control measures.     

Ultrasonographic appearance of exogenous isobutane gas in the mammary glands of dairy cows.

OBJECTIVE: To determine whether exogenous isobutane gas infused into the udders of dairy cattle could be detected ultrasonographically, and if so, what effects volume of gas infused and infusion pressure had on how long after infusion exogenous isobutane gas could be detected. DESIGN: Randomized block design. ANIMALS: 8 Holstein cows 28 to 32 days after parturition. PROCEDURE: In each cow, 1 mammary gland was not treated and the other 3 received 1 of 3 treatments by means of intramammary infusion: low volume-high pressure, low volume-low pressure, and high volume-high pressure infusion of isobutane gas. Mammary glands were examined ultrasonographically 1 hour before and 1, 3, 12, 24, 36, 48, 72, and 96 hours after treatment. RESULTS: After intramammary infusion of isobutane gas, bright echoes and associated acoustic shadows were seen ultrasonographically; echoes were no longer seen 72 hours after gas infusion. Percentages of mammary glands in which bright echoes were detected were not significantly different among the 3 treatment groups at any time during the study. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that exogenous isobutane gas infused into the mammary glands to enhance the appearance of the udder of show dairy cattle can be readily detected by ultrasonography.     

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.     

Persistence of uterine bacterial infection,and its associations with endometritis and ovarian function in postpartum dairy cows

We investigated the relationship between the persistence of uterine bacterial infections with cytologically determined endometritis and ovarian function in 65 postpartum Holstein cows. Vaginal mucus discharges were collected, and endometrial smear samples (n = 130) were collected for cytological and bacteriological examinations from the cows at weeks 5 and 7 postpartum (pp). Blood samples were collected at weeks 3, 5 and 7 pp to determine plasma progesterone concentrations to monitor ovarian activity. According to the bacteriological examination, cows were classified into four groups. The first group (n = 32; 49%) comprised cows negative for bacteria at weeks 5 and 7 pp. The second group (n = 11; 17%) comprised cows with bacterial infections at week 5 pp but that were clear of infection at week 7 pp. The third group (n = 12; 19%) comprised cows without bacteria at week 5 pp but that acquired an infection by week 7 pp. The fourth group (n = 10; 15%) comprised cows with bacterial infections at weeks 5 and 7 pp (persistence of infection). A positive correlation (P < 0.001) was noted between the severity of cytologically determined endometritis, purulent vaginal discharge and the persistence of infection. Cows with persistent infections had a significantly (P < 0.01) prolonged luteal phase compared with cows without infection. In conclusion, the prevalence of cytologically determined endometritis and prolonged luteal phase were significantly increased in cows with persistent infections.     

Body condition scores in dairy cows: associations with metabolic and endocrine changes in healthy dairy cows

Relationships between body condition scores (BCS), metabolic profiles and endocrine traits were investigated in 53 healthy Red Holstein cows. Cows were categorized into groups based on BCS ante-partum (a.p.: >3.25 or < 3.25) and on BCS losses during the first 8 weeks after calving (ABCS8 > 0.75 or < or = 0.75). Blood samples were collected 1 week before calving and every 2 weeks post-partum (p.p.). Cows with BCS a.p. >3.25 and deltaBCS < or = 0.75 were oldest and cows with BCS a.p. < or = 3.25 and deltaBCS < or = 0.75 were youngest. Cows with BCS > 3.25 a.p. and that lost > 0.75 BCS in the first 2 months of lactation exhibited signs of subclinical ketosis. If statistically adjusted for the effect of lactation number, average milk yield within the first 8 weeks p.p. and milk fat concentrations were similar between BCS groups, whereas milk protein concentrations differed significantly between BCS groups. Significant differences between groups were observed for blood plasma glucose, bilirubin, beta-hydroxybutyrate, non-esterified fatty acids and insulin concentrations. No differences were seen for albumin, urea, insulin-like growth factor-1, and 3.5.3'-triiodothyronine concentrations and for plasma activities of glutamate-oxalacetate transaminase, gamma-glutamyltransferase and glutamate dehydrogenase. There was a good agreement between BCS and profiles of metabolites and hormones related to energy metabolism in clinically healthy cows. Cows in good body condition a.p. had greater risks of metabolic problems because of excessive mobilization of body reserves. However, the metabolic status was best in cows with a BCS > 3.25 a.p.. if they did not lose much body condition p.p.     

Antifungal susceptibility of yeasts isolated from secrection of inflamed mammary glands in cows

Antifungal susceptibility of 150 strains of yeasts isolated from the infected mammary secretion of cows was determined. Their susceptibility to amphoterycin B, nystatin, flucytosine, tioconazole, ketoconazole, miconazole, fluconazole and clotrimazole was evaluated using the disc diffusion method on Yeast Nitrogen Base (YNB) and filter paper discs. The most active antifungal agents in vitro were found to be: tioconazole--96.7%, nystatin--95.4%, amphoterycin--94.0%, and flucytosine--92.7% of susceptible strains; the least active were fluconazole and clotrimazole--39.3% and 60.0% of susceptible strains, respectively. The individual species of yeasts were characterized by varied susceptibility to individual therapeutics.     

Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows

Staphylococcus chromogenes is a highly prevalent species in subclinical mastitis with a well-established impact on somatic cell count. Few data are available on its antimicrobial susceptibility. The objective of this study was three-fold: (1) to evaluate simple identification tests by comparing them with a genomic method; (2) to determine minimal inhibitory concentrations (MICs) of different antibiotics; (3) to search for the presence of important resistance mechanisms and resistance-determining genes.Seventy-three staphylococcal strains, all collected on different dairy farms, were tentatively identified as S. chromogenes based on their lack of hemolysis and their characteristic intermediate DNase activity. The identification of 70 strains was confirmed as S. chromogenes by tRNA intergenic spacer PCR (tRNA PCR). Three strains were identified as S. sciuri, a species that is naturally cloxacillin- and lincomycin-resistant.All 70 S. chromogenes strains were found to be normally susceptible to neomycin, gentamicin, erythromycin, enrofloxacin, and to penicillinase-stable penicillins and cephalosporins, represented in this study by cloxacillin. The latter result was confirmed by the absence of the mecA gene in each of 13 strains in which this gene was searched for. Twenty-seven (38%) strains were penicillinase producers. Three lincomycin-resistant S. chromogenes strains were found to carry the linA gene.It was concluded that S. chromogenes can be identified reliably in routine mastitis bacteriology, and that the only resistance of importance is against penicillinase-susceptible penicillins.     

Prevalence of parasite eggs and cysts in faeces from dairy cows in Colombia

Summary When 1, 711 bovine faecal samples from 113 farms in eight dairy areas of Colombia were examined for the presence of helminth eggsFasciola hepatica eggs were found in the faeces from 60% of the farms and samples from animals kept above 2,000 m. Strongyle eggs were found in faeces from 82% of the farms and in 18% of the samples.

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Resumen Se examinaron 1.711 muestras fecales provenientes de 113 fincas, localizadas en ocho zonas lecheras de Colombia. Se encontraron huevos deFasciola hapatica, en el 60% de fincas y muestras fecales de animales sobre los 2,000 m.s.n.m. Se encontraron tambien huevos de estrogilideos en heces del 82% de fincas y en el 18% de las muestras examinadas.

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Résumé La recherche d’oeufs d’helminthes sur 1711 échantillons de fécès provenant de 113 fermes de 8 districts laitiers de Colombie a permis de noter la présence d’oeufs deFasciola hepatica dans 60 p. 100 des fermes et échantillons originaires d’animaux vivant à plus de 2,000 m. Des oeufs de strongles ont été notés dans les fécès provenant de 82 p. 100 des fermes et 18 p. 100 des échantillons.

     

Characterization of herpesviruses isolated from lactating dairy cows with mammary pustular dermatitis.

Herpesviruses serologically indistinguishable from the DN599 strain of bovine herpesvirus were isolated from 2 separate enzootics of mammary pustular dermatitis in lactating Holstein-Friesian cows.     

Enzymatic activity of yeasts isolated from the inflamed mammary secretion in dairy cows

The aim of the study was to evaluate enzymatic activities of yeasts isolated from inflammatory mammary secretion. The yeasts isolated from cows with clinical and sub-clinical mastitis (134 strains) included: Candida krusei (62 strains), Candida kefyr (48 strains), Candida lusitaniae (17 strains) and Candida famata (7 strains). The API ZYM system was used containing substrates to assess 19 hydrolytic enzymes. Substantial differences in the number and activity of hydrolyses were demonstrated in individual species. In Candida krusei, acid phosphatase showed the highest activity (4.36 points), in Candida kefyr and Candida lusitaniae--leucine arylamidase (4.93 and 4.25 points, respectively), in Candida famata--alpha-glucosidase (4.75 points). No activity of trypsin, chymotrypsin, alpha-galactosidase, beta-glucuronidase, alpha-mannosidase or alpha-fucosidase was observed in any of the yeasts examined.     

Prevalence of Giardia duodenalis genotypes in adult dairy cows

The prevalence of Giardia duodenalis genotypes was determined in adult dairy cows. Fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. The prevalence of G. duodenalis infection, ranged from 3% to 64%, with an average prevalence of 27% (144 positive cows out of 541 examined). DNA sequence analysis of the 16S rRNA gene revealed the presence of both Assemblage A and Assemblage E, G. duodenalis. Overall, Assemblage E was present in 25% of all animals tested and Assemblage A was present in 2% of the animals. As a percentage of G. duodenalis isolates, Assemblage E represented 94% and Assemblage A represented 6%. Although, most of the cows were infected with a genotype that is not known to be infectious for humans, adult cows on five farms did harbor varying levels of zoonotic Assemblage A, G. duodenalis. Therefore, although adult cows do not appear to be a significant source of human infectious cysts in the environment, the risk from this age group should not entirely be discounted.     

奶牛乳腺组织亚细胞组分的双向凝胶电泳分析

利用超速离心结合梯度离心法分离亚细胞组分的技术路线,以提高奶牛乳腺组织蛋白双向凝胶电泳的分离效率。奶牛乳腺组织液氮研磨破碎后,差速离心分离成细胞核、线粒体、高尔基体、溶酶体4个亚细胞组分,Nyco-denz纯化,2-DE分离蛋白,PDQUest8.0软件分析凝胶图谱。结果显示,奶牛乳腺组织细胞经亚细胞分离后,电泳分辨率大大提高,特别是细胞核蛋白质检出效率明显提高。不同亚细胞蛋白质组电泳图谱的差异显示了其蛋白质组构成的不同。由此可见超速离心结合梯度离心是一种有效的奶牛乳腺亚细胞组分分离手段,亚细胞蛋白质组学克服了蛋白质组的一些缺陷并可将蛋白质组研究引向亚细胞水平。     

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.     

Gram-negative bacterial infections of the mammary gland in cows

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were less than 28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P less than 0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.     

Natural antibodies related to metabolic and mammary health in dairy cows

Natural antibodies (NAb) are defined as antibodies that circulate in normal healthy individuals under the absence of deliberate antigenic stimulation. Two types of NAb are distinguished: NAb towards exogenous antigens and NAb towards autoantigens (N(A)Ab). The objectives of the current study were threefold. First, we studied the relation between metabolic health and concentrations of NAb binding keyhole limpet hemocyanin (KLH) or lipopolysaccharide (LPS) in milk and plasma of dairy cows in early lactation. Second, we determined the presence of N(A)Ab binding transferrin, myosin and thyroglobulin in bovine milk. Third, we studied the relation between N(A)Ab in bovine milk and mammary health. For the first objective, dairy cows were either fed a control (C) (n = 8) or a diet where 2 kg of concentrates were replaced by an iso-energetic concentrate containing marine algae (ALG) from week −3 till 8 postpartum (experiment 1). Plasma and milk samples were analyzed weekly for NAb binding either KLH or LPS. Plasma was analyzed for glucose, non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA). For the second and third objective, milk samples were collected weekly from 96 dairy cows from week 2 till 9 postpartum and analyzed for milk composition and N(A)Ab binding myosin, transferrin and thyroglobulin (experiment 2). For both datasets, N(A)Ab titers are expressed as ²log values of the highest dilution giving a positive reaction. Data are expressed as means ± SEM. Repeated observations were analyzed in a mixed model. In experiment 1, no diet effect (P > 0.05) was observed on NAb binding LPS in plasma or milk, NAb binding KLH in milk was greater (P = 0.05) for cows fed the control diet. Concentration of NAb binding KLH and LPS in plasma was negatively related to plasma NEFA concentration (P < 0.05). In experiment 2, NAb binding myosin (5.66 ± 0.06), thyroglobulin (4.85 ± 0.06), and transferrin (5.76 ± 0.07) were identified in milk. Clinical mastitis incidence (9%) tended to be positively related to concentration of NAb binding myosin (P = 0.06) and negatively related to Nab binding transferrin (P = 0.08). In conclusion, NAb binding KLH and LPS in plasma and milk are related to metabolic health, as indicated by plasma NEFA concentration. Furthermore, this study demonstrates the presence of N(A)Ab in bovine milk and shows trends for a relation between N(A)Ab binding auto-antigens and mastitis. Future studies should confirm these trends and shed light on the predictive value of N(A)Ab in bovine milk for mammary health.     

Staphylococcus aureus isolates from dairy cows and humans on a farm differ in coagulase genotype

Staphylococcus aureus is a frequent cause of animal and human infections. The aim of the present study was to test diversity of the populations of S. aureus colonising cattle and humans sharing an infected environment. Eighty-six S. aureus isolates obtained from dairy cows, from people coming into contact with dairy cows on the farm and the other farm personnel were characterized by restriction fragment length polymorphism of the coagulase gene. Molecular analyses identified ten polymorphism types with prevalent presentation of type II in isolates from cow's milk and type IV in isolates from people coming into contact with dairy cows on the farm (the cattlemen) and the other farm personnel. Seven further genotypes were identified among the isolates from the cattlemen. The results indicate that the strains dominating in human population did not equate to the causative agents of bovine mastitis.     

Analyses of factors affecting dry matter intake of lactating dairy cows

An experiment was conducted to analyze feed, climate and animal factors affecting dry matter intake (DMI) in lactating dairy cows. Sixteen lactating Holstein cows, with parity from 1 to 6, were assigned to a feeding trial for 2 years, comprising 31 lactations. The animals were fed Italian ryegrass silage, oat hay, alfalfa hay, beet pulp and three types of concentrate. The data, pooled and classified by stage of lactation, season of lactation and parity were analyzed by stepwise multiple regression to determine the nature and extent of factors affecting DMI. A total of 45 prediction equations for DMI were derived. Energy‐corrected milk yield or milk yield was selected as the primary factor of DMI in all the equations in which the ratio of contribution (R²) varied from 0.26 to 0.67. The dietary concentration of organic cell wall, crude fiber, crude protein, organic b fraction, forage to concentrate ratio, average ambient temperature and temperature–humidity index were selected as the secondary factors affecting DMI for pooled data, late lactation (251–350 days of lactation), summer (June–August), spring (March–May), ≥4th lactation, autumn (September–November) and 3rd lactation, respectively, and improved R² up to 0.77. Except for an impact of bodyweight in several equations, feed and climatic factors significantly improved prediction equations effectively for data classified in different ways. To estimate DMI accurately in lactating dairy cows, feed and climatic factors should be considered for specific conditions.     

The elimination of serum-resistant Escherichia coli from experimentally infected single mammary glands of healthy cows.

Neutrophils isolated from mammary glands stimulated with a staphylococcal culture filtrate efficiently killed serum-resistant strains of Escherichia coli. This study was extended and it was shown that an infusion of wide ranging numbers (5 X 10(1) to 5 X 10(6)) of the same strains of E coli into a single mammary gland resulted in bacterial growth, which was eliminated following neutrophil infiltration. This elimination occurred before the appearance of any clinical signs. Once bacterial kill had started in the gland, it continued in the milk after withdrawal from the gland. These results offer an explanation of why causative microbial agents cannot be isolated from some cases of clinical mastitis.     

Prevalence and interrelationships of hoof lesions and lameness in Swedish dairy cows

The prevalence of hoof lesions and lameness in 4899 heifers and cows was determined at claw trimming one time in a cross-sectional study on 101 Swedish dairy farms, 1996-1998. The percentage of affected animals was 41% for heel-horn erosion, 30% for sole haemorrhages, 27% for erosive dermatitis, 21% for abnormal claw shape, 14% for white-line haemorrhages, 8.8% for white-line fissures, 8.6% for sole ulcers, 3.3% for double soles, 2.3% for verrucose dermatitis, and 1.8% for interdigital hyperplasia (IH). Seventy-two percent of all animals had at least one hoof lesion. The prevalence of lameness was 5.1%; most hoof lesions did not cause lameness. Differences between herds were substantial; the herd-specific, animal-level prevalence of lesions ranged from 25 to 98% and of lameness from 0 to 33%. Sole haemorrhages were found in all herds. The proportion of variance at the between-herd level was particularly high for heel-horn erosion (62%) and abnormal claw shape (54%). Strong correlations between lesions were found within hooves (and animals), e.g. for heel-horn erosion and dermatitis (Spearman's rank correlation, r(s)=0.36 and 0.37, respectively) and for sole and white-line haemorrhages (r(s)=0.25 and 0.28). Most hoof lesions affected hind and front hooves bilaterally, whereas the correlation between hind and front hooves generally was lower. Herds that ranked high for prevalence of sole ulcer also ranked high for sole haemorrhages and for abnormal claw shape and herds that ranked high for dermatitis also ranked high for heel-horn erosion, verrucose dermatitis and IH. Abnormal claw shape was strongly associated with sole ulcer (r(s)=0.41 at cow level)-suggesting the importance of maintaining a correct claw shape for the prevention of hoof-horn lesions.